The objective of the present invention relates to a diagnostic method for detecting the presence or absence of exogenous polynucleotides from viruses, rickettsias, bacteria and the like as the etiology of diseases or a polynucleotide carrying specific genetic information in organisms; a polynucleotide preparative method for polynucleotide structural analysis; and an immobilized probe chip used therefor.
Japanese Patent Laid-open No. 58-31998 discloses a method for capturing a target polynucleotide (DNA or RNA) sample on a solid phase as well as a method for detecting a target exogenous polynucleotide from bacteria, viruses and the like in blood and excretion samples. According to such methods, the polynucleotide in a sample is denatured by heating and the like into single strands, which is then immobilized on nitrocellulose membrane. After reacting the membrane with a polynucleotide probe being labeled with a radioisotope and having a nucleotide sequence complementary to the sequence of the polynucleotide of a detecting bacterium or a virus, the membrane is washed. If the polynucleotide of a bacterium or a virus is contained in the sample, the labeled polynucleotide probe hybridizes with the polynucleotide, and thus remains on the membrane. Detection of the associated product by autoradiography enables the determination as to whether or not the target polynucleotide is present.
S. R. Rasmussen et al. describe another method for capturing a target polynucleotide (DNA or RNA) sample on a solid phase in Analytical Biochemistry 198, 128-142(1991). According to the method, the phosphate group at the 5' terminus of a polynucleotide is activated by using 1-methylimidazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Then, the polynucleotide is immobilized onto a polystyrene microplate having a secondary amine on the surface thereof. According to the method, the activated 5' terminal phosphate group reacts with the secondary amine, so that the 5' terminal of the polynucleotide is covalently immobilized onto the microplate surface. In this example, a target oligonucleotide in a sample can be captured by using an immobilized polynucleotide. A specific oligonucleotide can be detected using a .sup.32 P-labeled probe according to any of the methods.